Knockout mouse production assisted by Blm knockdown

To elucidate roles of any particular gene or genetic element in higher-order biological processes, a general method(s) for genome editing and creation of such genome-modified species is essential. Therefore, the approach of genome editing in mouse embryonic stem cells (ESCs) and ESC-based genome-modified animal production is commonly used. For gene targeting via homologous recombination (HR), a targeting vector possessing a drug-resistant gene plus 5’ and 3’ homology sequence arms is introduced into the cells. Although there is a well-established general protocol and many genes are already targeted by this method in ESCs, the targeting efficiency varies at different target loci and sometimes is too low. Recently, other genome editing technologies such as zinc finger nuclease (ZFN) [1, 2], TAL effector nuclease (TALEN) [3, 4] and CRISPR/Cas9 [5, 6] systems have been developed. If specific and highly competent nuclease can be designed, all these systems would work well for gene targeting in mammals [7, 8]. The CRISPR/Cas9 system is the newest of the systems, but it is extremely useful. Unlike the ZFN and TALEN systems, the CRISPR/Cas9 system uses RNA as a guide molecule and a 20-nt RNA sequence specifies the target DNA site. Therefore, preparation of specific targeting materials is much easier and simpler. Furthermore, if Cas9 mRNA is delivered along with guide RNA into mouse zygotes, genome-edited mice can be easily obtained. However, even for the CRISPR/Cas9 system, some technical challenges still exist. An obvious one is the off-target mutagenesis risk due to the 20-nt sequence restriction of the target specificity. Furthermore, because homology-directed repair (HDR) is less efficient in mammals, targeted gene replacement or insertion mediated by HDR is inefficient and the zygote injection method with Cas9 RNA, guide RNA and a targeting template construct is generally not practical for creating such genome-edited mice. For genome editing using a standard targeting vector, various trials have been applied to improve gene targeting frequencies by HR [9]. Among them, knockdown of the Bloom syndrome gene, BLM, has been shown to enhance gene targeting efficiencies in various human cell lines [10]. BLM encodes RecQ type DNA helicase [11] and plays a role in the suppression of HR [12]. However, it has not yet been investigated whether this approach in ESCs is applicable for knockout mouse production. Therefore, in this study, we targeted multiple different gene loci with or without Blm knockdown in ESCs and used the targeted ESC clones obtained with Blm knockdown for chimeric mouse production and germline transmission. For Blm knockdown in ESCs, we designed three different Blm siRNAs (siBlm1-3). At 48 h post transfection, the amount of Blm mRNA was significantly decreased in ESCs transfected with all independent Blm siRNAs (Fig. 1a). Western blot analysis also showed significant reduction of the Blm protein amount specifically by Blm siRNA treatment (Fig. 1b). Among them, siBlm-2 and siBlm-3 induced higher knockdown efficiency than siBlm-1. Therefore, we selected siBlm-2 and siBlm-3 and combined them for further Blm knockdown experiments (Fig. 1c). Fig. 1. Blm was knocked down by siRNAs. a) 20 nM of each Blm siRNA was transfected into KY1.1. At 48 h after transfection, the Blm mRNA level was measured by quantitative RT-PCR using two primer sets. b) The expression level of Blm protein was determined by western blot. ... To validate how Blm knockdown affects gene targeting efficiency in ESCs, we targeted three different gene loci, namely, Prdm5 on chromosome 6, Prdm8 on chromosome 5 and, Arl14ep on chromosome 2, with or without Blm siRNAs pretreatment. Prdm5 and Arl14ep are expressed but Prdm8 is silent in ESCs (not shown). We used standard gene targeting vectors for these three genes (Fig. 2a) [13]. Forty-eight hours before transfection of the targeting vectors, one part of the cells was transfected with Blm siRNAs in the condition shown in Fig. 1c. Then, the ESCs transfected with each targeting vector were selected with G418. More than 200 drug-resistant colonies per transfection were screened for proper gene targeting. As summarized in Table 1, the targeting efficiencies of Prdm5, Prdm8 and Arl14ep were 8/214 (3.7%), 8/796 (1.0%) and 14/240 (5.8%) for ESCs without Blm knockdown, respectively. For the ones with Blm knockdown, the targeting efficiency was 29/232 (12.5%) for Prdm5, 15/363 (4.1%) for Prdm8 and 32/240 (13.3%) for Arl14ep. Thus, pretreatment with the Blm siRNAs pre-treatment enhanced the targeting efficiency for all three gene loci, and the fold activation enrichment was 3.4 for Prdm5, 4.1 for Prdm8 and 2.3 for Arl14ep. In another experiment for Arl14ep gene targeting, we screened cells transfected with control siRNA (siC-L) in addition to cells treated with Blm siRNAs (Table 2). This time, the targeting efficiencies were generally low but treatment with the Blm siRNAs gave higher fold activation enrichment than that with siC-L (2.6 for Blm siRNAs and 1.4 for siC-L) suggesting that the effect of Blm siRNAs is not non-specific. Fig. 2. Schematic diagram for gene targeting. a) Prdm5 targeting: lox P (shaded triangle)-frt (open triangle)-PGK-Neo-frt and another lox P site were introduced into upstream and downstream of exon (Ex) 1, respectively. Prdim8 targeting: lox P-frt-PGK-Neo-frt ... Table 1. Gene targeting efficiency with or without Blm knockdown Table 2. Influence of siRNA on gene targeting Then, we examined whether the targeted ESC clones obtained with Blm siRNA pretreatment maintained pluripotency, especially for the germline transmission potential. Since sister chromatid exchanges (SCEs) are highly increased in Blm knockout or knockdown cells [14,15,16,17,18,19], we first checked chromosome stability. As shown in Table 3, we performed a karyotype analysis of three targeted ESC clones for Arl14ep (#12, #13 and #27) with Blm knockdown and a parental ESC, KY1.1, as a control. For all targeted clones examined, the average chromosome number per cell was not changed and remained at ~40. Table 3. Karyotype analysis of the established targeted ESC lines with Blm knockdown Finally, we created chimeric mice using the targeted ESC clones obtained with Blm knockdown for the three genes, Prdm5, Prdm8 and Arl14ep. As summarized in Table 4, 2/3 to 4/4 of them generated > 80% chimeric mice as judged by the coat color contribution. Furthermore, germline transmission of the targeted allele was confirmed for all three gene loci from those good chimeric mice (more than two lines for each gene). Therefore, we concluded that Blm siRNA pretreatment does not have clear negative effects on chromosomal stability or germline transmission potential of the obtained targeted ESC clones. Table 4. Production of chimeric mice and germline transmission by the targeted ESC lines with Blm knockdown In conclusion, Blm knockdown provides a general benefit for efficient ESC-based and HR-mediated knockout mouse production.